Polyketides and Their Synthesis and Use

ABSTRACT

The rapamycin gene cluster is an example of a gene cluster which includes a gene (rapL) leading to the formation of a precursor compound (pipecolic acid, in this case) which is required for inclusion in the larger product (rapamycin) produced by the enzymes encoded by the cluster. We have produced a mutant strain containing a rapamycin gene cluster in which the rapL gene is disabled. The strain does not naturally produce rapamycin but does so if fed with pipecolic acid. By feeding with alternative carboxylic acids we have produced variants of rapamycins. Tests have shown biological activity.

The present invention relates to polyketides and their synthesis anduse. It is particularly, but not exclusively, concerned with variants ofrapamycin.

Rapamycin (see FIG. 2) is a lipophilic macrolide, of molecular weight914, with a 1,2,3-tricarbonyl moiety linked to a pipecolic acid lactone.Sequencing of the putative biosynthetic genes of rapamycin has revealedthe presence of three exceptionally large open reading frames encodingthe modular polyketide synthase (Schwecke et al., P.N.A.S. 92 (17)7839-7843 (1995)). On either side of these very large genes are rangedopen reading frames which appear to encode enzymes that would berequired for rapamycin biosynthesis.

The cluster also contains a novel gene (rapL) whose product is proposedto catalyse the formation of the rapamycin precursor L-pipecolate (2)through the cyclodeamination of L-lysine (1) (Molnar et al., Gene 169,1-7 (1996)):—

The biosynthesis of rapamycin requires a modular polyketide synthase,which uses a shikimate-derived starter unit and which carries out atotal of fourteen successive cycles of polyketide chain elongation thatresemble the steps in fatty acid biosynthesis. L-pipecolic acid is thenincorporated into the chain, followed by closure of the macrocyclicring, and both these steps are believed to be catalyzed by apipecolate-incorporating enzyme (PIE), the product of the rapP gene.Further site-specific oxidations and O-methylation steps are thenrequired to produce rapamycin.

We have now found that we can genetically engineer an S. hygroscopicusorganism in which the (rapL) gene is inactivated. The organism cannotproduce rapamycin under normal growth conditions but can do so if fedpipecolate. Furthermore feeding the mutant organism with differentsubstrates leads to the production of variants of rapamycin. The samegeneral method can be applied to other systems which involve a precursorcompound which is produced by a gene product, e.g. the very closelyrelated FK506 and immunomycin systems which also involve pipecolate.

Thus according to the present invention in a first aspect there isprovided a process of modifying a gene cluster involved in thebiosynthesis of a polyketide, said gene cluster including a gene (“theprecursor gene”) responsible for the production of an enzyme which isresponsible for the production of a precursor compound which isincorporated into said polyketide; said process comprising the step ofdeleting or inactivating said precursor gene. Suitably said process ofdeleting or inactivating said precursor gene employs phage-mediated genereplacement. In preferred embodiments of the invention the gene clusteris the gene cluster for the production of rapamycin in S. hygroscopicusand the precursor gene is the rapL gene whose product is responsible forthe production of L-pipecolate.

In a second aspect the invention provides a process for producing apolyketide comprising modifying a gene cluster by the process accordingto the first aspect and expressing the modified gene cluster in thepresence of a variant precursor compound which is incorporated so that avariant polyketide is produced. For the rapamycin system, examples ofthe variant precursor compound include L-proline,L-trans-4-hydroxyproline, L-cis-4-hydroxyproline, L-cis-3-hydroxyprolineand trans-3-aza-bicyclo[3,1,0]hexane-2-carboxylic acid.

In further aspects the invention provides polyketides as producible bythe above method, pharmaceuticals comprising such polyketides, and theuse of such polyketides in preparing pharmaceutical compositions, e.g.immunosuppressants containing rapamycin variants.

Some embodiments of the invention will now be described in greaterdetail with reference to the accompanying drawings in which;

FIG. 1 shows a portion of the rapamycin gene cluster, wild type andmutated, and the phage vector used to perform mutation;

FIG. 2 shows structures of rapamycin and some variants; and

FIGS. 3 and 4 illustrate the effects of rapamycin and variants on humanlymphoblastoid cell lines.

In order to facilitate the production of variant rapamycins, achromosomal mutant of S. hygroscopicus was created by phageφC31-mediated gene replacement using the method of Lomovskaya et al.[Microbiology (UK) 1997, 143, 815-883]. A unique BamH I site was found42 bp into the rapL gene (1032 bp long). This BamH I site was removed byend-filling with E. coli DNA polymerase I thus creating a frameshift inthe rapL gene. A 3 kb EcoR I fragment encompassing the entire rapL geneflanked by rapK and part of the rapM genes respectively was cloned intothe phase vector, KC515. The recombinant phage was used to transfect S.hygroscopicus. A double recombination event resulted in the creation ofa chromosomal mutant of S. hygroscopicus with a frameshift in rapL. Thisis summarised in FIG. 1.

Materials and Methods

Note: the reader is also referred to L. E. Khaw et al., J. Bacteriol.,180 (4) 809-814 (1998) which is incorporated herein by reference, forboth experimental details and discussion of the work and the backgroundthereto.

Materials. All molecular biology enzymes and reagents were fromcommercial sources. Viomycin was a gift from Pfizer, L-pipecolic acid,L-proline, 3,4-dehydroproline, picolinic acid, pyrrole-2-carboxylicacid, trans 4-hydroxyproline, c is 4-hydroxyproline, c is3-hydroxyproline and (±)-trans-3-aza-bicyclo[3,1,0]hexane-2-carboxylicacid were obtained from Aldrich Chemical Company.

Bacterial Strains, Phages and Growth Conditions

Escherichia coli DH10B (GibcoBRL) was grown in 2×(tryptone-yeastextract) medium as described by Sambrook et al, (“Molecular Cloning”,Cold Spring Harbor (1989)). Vector pUC18 was obtained from New EnglandBiolabs. or Sigma Chemical Co. E. coli transformants were selected with100 mg/ml ampicillin. The rapamycin producer Streptomyces hygroscopicusNRRL 5491 (from ATCC) and its derivatives were maintained on SY agar(Soluble starch 1.5%; yeast extract 0.1%; K₂HPO₄ 0.1%; MgSO₄×7H₂O 0.1%;NaCl 0.3%; N-tri(Hydroxymethyl)methyl-2-aminoethanesulfonic acid (Tes)buffer 30 mM, pH7.4; agar 1.5%), and cultivated in Tryptic Soy Brothwith 1.0% glucose, 100 mM MES pH6.0, supplemented with 10 ug/ml viomycinwhen required. S. lividans J11326 (D A Hopwood et al: “GeneticManipulation of Streptomyces: a laboratory manual”, The John InnesFoundation, Norwich, England (1985)) was cultivated in YEME (Hopwood etal., 1985) or Tap Water Medium (0.5% glucose; 1% sucrose; 0.5% tryptone;0.25% yeast extract; 36 mg EDTA; pH 7.1). Liquid cultures were grown at30° C. in Erlenmeyer flasks with shaking at 200-250 rpm. Infection withthe atr actinophage KC515 (Hopwood (1985) op. cit. and K. F. Chater in:“The Bacteria” IX (119-158), New York 1986) and its derivative ΦΔrapL(present work) were done on solid DNA medium supplemented with 10 mMMgSO₄, 8 mM Ca(NO₃) and 0.5% glucose (Hopwood et al., 1985).

Isolation and In Vitro Manipulation of DNA

DNA manipulations, PCR and electroporation procedures were carried outas described in Sambrook et al (1989). Total S. hygroscopicus DNA wasisolated using the Gibco genomic DNA isolation kit. Southernhybridizations were carried out with probes labelled with digoxigeninusing the DIG DNA labelling kit (Boehringer Mannheim). DNA fragments forlabelling and subcloning were isolated with the Qiaex (Qiagen) gelextraction kit.

Construction of ΦΔrapL Carrying a Frameshift in the rapL Gene forHomologous Recombination in S. hygroscopicus

pUC3EcoRI was constructed by cloning a 3034 bp Eco RI fragment(nucleotides 93956 to 96990 of the rap cluster) (T. Schwecke et al.,P.N.A.S. 92, 7839-7843 (1995)) encompassing the entire rapL gene flankedby rapK and part of the rapM genes respectively into an Eco RI-cut pUC18modified vector where the Bam HI site in the polylinker region has beenremoved. A unique Bam HI site (starting at nucleotide 95036 of the rapcluster) was found 42 bp into the rapL gene (nucleotide 95078 to 94047of the rap cluster; 1032 bp long). Plasmid pUC3Eco RI was digested withBam HI and the cohesive ends were filled in by treating it with E. coliDNA polymerase I (Klenow fragment). The ligated plasmid DNA wasredigested with Bam HI and used to transform E. coli. Ampicillinresistant transformants were selected and their plasmid DNA checked forthe removal of the Bam HI site by restriction enzyme analysis. This wasconfirmed by DNA sequencing. The 3 kb insert was excised from theplasmid with Eco RI and the cohesive ends blunt-ended by treatment withE. coli DNA polymerase I (Klenow fragment). The blunt-ended insert wascloned into Pvu II cut phage vector KC515, resulting in ΦΔrapL.

Protoplasts of S. lividans J11326 were transfected with the phageconstruct as described by Hopwood et al. (1985). Recombinant phage wasidentified using PCR analysis. Infection of S. hygroscopicus NRRL 5491with ΦΔrapL was done according to Lomovskaya et al (Microbiology. 143,875-883 (1997)) on DNA plates supplemented with glucose, MgSO₄ andCa(NO₃). Lysogens were selected by overlaying the plates with 50 μg ml⁻¹(final concentration) viomycin 24 h post-infection. Strains that hadundergone a second recombination event deleting the integrated phagewere identified by selecting viomycin sensitive isolates after threerounds of non-selective growth and sporulation on SY plates. Theinsertion and subsequent loss of the phage were confirmed by genomicSouthern hybridizations.

Precursor Feeding and Fermentation of S. hygroscopicus ΔRapL

Precursor feeding of S. hygroscopicus ΔRapL was performed routinely in500 ml flasks containing 10 ml of Tryptic Soy Broth with 1.0% glucose,100 mM MES pH6.0, supplemented with the appropriate pipecolic acidanalogue, at a final concentration of 1 mg/ml. S. hygroscopicus ΔRapLwas also cultivated in 2 l flasks containing 400 ml ofchemically-defined media as described by Cheng et al (Appl. Microbiol.Biotechnol. 43, 1096-1098, (1995)). For large scale fermentation, 10 μlof spores of S. hygroscopicus ΔRapL was used to inoculate a 100 ml flaskcontaining 30 ml of Tryptic Soy Broth medium. The flask was incubated ona rotary shaker (300 rpm) at 28° C. for 4 days. 4 ml of the first seedculture was transferred to a 2 l flask (second seed culture) containing400 ml of the medium and incubated on a rotary shaker (300 rpm) at 28°C. for 4 days. The second seed culture was transferred to a 201fermenter containing 15 l of the medium. Trans 4-hydroxyproline wasadded to the medium aseptically to a final concentration of 1 mg/ml. Thefermentation was carried out at 28° C. for 4 days, with an agitationrate of 500 rpm. The cells were harvested and extracted with twice theirvolume of methanol overnight.

Purification and Analysis of Rapamycin and its Derivatives

After 3-4 days fermentation mycelia were collected by filtration andextracted with two volumes of methanol at room temperature for 1 h. Thecrude extracts were analysed by lc-ms using a Finnigan MAT (San Jose,Calif.) LCQ with a Hewlett-Packard 1100 HPLC. The large scalefermentation was worked up similarly. The crude extract was evaporatedto dryness and then purified by flash chromatography (Merck silica gel60, no. 9385) with acetone/hexane 1/1. The fractions containingrapamycins were further purified by preparative HPLC on a 250×20 mm RP18column (HPLC Technology, Macclesfield, UK) using standard conditions.The 15 l fermentation yielded about 15 mg of pure prolyl-rapamycin and 3mg of 4-hydroxy-prolyl-26-demethoxy-rapamycin. NMR spectra weredetermined on a Bruker DRX 500 spectrometer.

Biological Activity of Rapamycin Analogues

Rapamycin induces a specific cell cycle arrest in G1 in the cell line536, which is a human B lymphocytic line immortalised by Epstein Barrvirus infection. The potency of each analogue was compared to that ofrapamycin using the 536 cells as a bioassay. The 536 cells (obtainedfrom the human genetic mutant cell repository, Camden, N.J., USA) werecultured in Iscoves medium supplemented with 10% fetal calf serum. Forbioassay, 536 cells were seeded into 96 well microtitre plates at 10,000per well in 100 μl of growth medium. Drug stocks of 1 mM in DMSO wereprepared and further dilutions were made to give a constant finalconcentration of 0.1% DMSO in growth medium. Control cultures weretreated with 0.1% DMSO in growth medium; experimental cultures receiveda final concentration of 10⁻⁷M, 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰ M rapamycin orrapamycin analogue. Each culture was set up in triplicate and replicateplates were labelled with 1 μCi tritiated thymidine (AmershamInternational, specific activity 70 Ci/mM) per well for 3 h at either 0h; 24 h; or 48 h incubation with drugs. At the respective time pointsthe cultures were harvested onto glass fibre paper to trap the DNAfollowing water lysis; free nucleotides were washed away. Radioactivityincorporated into the filter discs/trapped DNA was counted in a Packardscintillation counter using biodegradable scintillation fluid.

Results

Characterisation of a Frameshift Chromosomal Mutation in the rapL Gene

To confirm that the rapL gene product is indeed involved in thebiosynthesis of rapamycin as a precursor feeder, the frameshiftchromosomal mutant S. hygroscopicus ΔRapL was isolated as described inMaterials and Methods. This mutation was investigated by Southern blothybridization using the 3 kb EcoRI fragment (93956-96990) to probe BglII/Bam HI digested chromosomal DNA. Analysis of the wild-type S.hygroscopicus shows the expected 5.9 kb (representing nucleotides89118-95036) and 2.7 kb Bam HI/Bgl II fragments (representingnucleotides 95036-97710) after hybridisation. When chromosomal DNA of S.hygroscopicus ΔRapL was treated similarly, only a 8.6 kb Bam HI/Bgl IIfragment (representing nucleotides 89118-97710) was detected, indicatingthat the Bam HI site at position 95036 has been removed. This wasconfirmed by PCR analysis. Chromosomal DNA was subjected to PCR usingoligonucleotide primers identical to, respectively, the sequences fromnucleotide 93950 to 93968; and from 96990 to 97010. The expected 3 kbDNA fragment was amplified from wild type DNA and, following BamIIIdigest, two bands roughly 2 kb and 1 kb in size were detected. Insamples containing S. hygroscopicus ΔRapL chromosomal DNA the 3 kb PCRproduct amplified was found to be resistant to BamHI digestion.

Precursor Feeding of the Chromosomal Mutant S. hygroscopicus ΔRapL

Growing cultures of the mutant S. hygroscopicus ΔRapL were fed withdifferent amino acid precursors (table 1). Only the three prolinederivatives were found to be incorporated as judged by LC-MS. The mainrapamycin derivative in the fermentations apart from prolyl rapamycin isa compound with m/z 908 which could correspond to a hydroxy-rapamycinlacking a methoxy group. Smaller amounts of a compound with m/z 938 werealso detected which would correspond to hydroxy-prolyl-rapamycin.MS-fragmentation experiments as well as the characteristic UV spectraclearly indicated that these compounds are rapamycin derivatives with ahydroxyproline incorporated. In order to get enough material for NMRcharacterisation we fed hydroxyproline on a large scale to the mutant(15 L broth) and isolated 3 mg of the compound with m/z 908 as describedin material and methods. The NMR data (table 2) showed the chemicalshifts and couplings expected for the hydroxy-proline spin system. Thechanged chemical shifts for the positions 26 and 27 and the unchangedshifts for positions 38-40 as compared to rapamycin proved that themethoxy group is missing at position 26. MS-fragmentation data (table 3)confirmed these findings. This can be inferred from the loss of theC15-C26-fragment leading to a fragment with m/z 644 for both of the newrapamycin derivatives. Furthermore, the loss of the C28-C42-fragment(322 amu) can be seen for both compounds as well as for rapamycin,indicating that there is no modification in this part of the molecules.The ions at m/z 807 and 777 respectively which are equivalent to theloss of the amino acid (131 amu) confirm the presence of OH-proline.This means that the compound with m/z 938 is 4-hydroxyproplyl-rapamycin.

TABLE 1 Retention Mass time (m/z. LC-MS Main Compound fed Incorporation(M + Na⁻) (min) Product L-pipecolic Yes 936 8.84 rapamycin acidL-proline Yes 922 7.99 prolyl- rapamycin L-trans-4- Yes 938/9085.35/6.29 4-hydroxy- hydroxy prolyl- proline rapamycin and 4- hydroxy-prolyl-26- demethoxy- rapamycin L-cis-4- Yes 938/908 5.35/6.29 as abovehydroxyproline L-cis-3- Yes 938/908 5.35/6.29 3-hydroxy- hydroxyprolineprolyl- rapamycin and 3- hydroxy- prolyl-26- demethoxy- rapamycinpicolinic No acid pyrrole-2- No carboxylic acid

TABLE 2 Position ¹H8 (ppm) ¹³C8 (ppm) 1 171.30  2 5.24 58.17 3 2.65,1.69 38.48 4 4.38 70.63 5 3.37, 2.94 56.53 26 3.58 not determined 273.89 70.63 38 2.93 83.90 39 3.37 73.95 40 1.99, 1.33 31.22 49 3.12 55.6851 3.39 56.50

TABLE 3 4- 4-hydroxyprolyl-26- Rapamycin hydroxyprolylrapamycindemethoxyrapamycin m/z m/z m/z 936 938 908 904 906 876 807 807 777 (lossof (los of (loss of pipecolate, hydroxyproline, hydroxyproline, 129 amu)131 amu) 131 amu) 642 644 644 614 616 586 596 598 568 582 584 554 564566 536

Preparation of trans-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acidrapamycin

A 2 L fermentation of S. hygroscopicus ΔRapL fed with(+/−)-trans-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid (0.5 mg ml⁻¹)was grown for 5 days in TSBGM medium (Khaw et al., (1998) J. Bacteriol.180, 809-814.). The cells were collected by filtration and extractedwith 1 L of methanol at 4° C. overnight. High pressure liquidchromatograph-electrospray ionization mass spectrometry (HPLC-ESIMS)analysis of the crude methanol extract was performed at this stage usinga Hewlett-Packard 1100 LC attached to a Finnigan-Mat LCQ massspectrometer. Trans-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acidrapamycin was detected in the fermentation broth.

The methanol extracts were combined and concentrated under reducedpressure. The aqueous residue was diluted with 500 mL of distilledwater, and extracted three times with 500 mL of distilled ethyl acetate.The combined ethyl acetate extracts were dried with anhydrous sodiumsulphate, and evaporated to dryness. The resulting yellow residue waspurified by flash column chromatography on a 150 mm×30 mm (diameter)silica gel column [Merck 60] eluted isocratically with a 1:1 (v/v)mixture of acetone/hexane.

The fractions were analysed by electrospray mass spectrometry. MS-MS andMs^(n) were used to determine the structure of the new rapamycin in thefractions from the flash silica column.

The fractions containing trans-3-aza-bicyclo[3.1.0]hexane-2-carboxylicacid rapamycin were further purified by reversed-phase preparative HPLCon a 250×20 mm (diameter) Prodigy ODS3 column (Phenomenex) usinggradient elution starting at 70/30 (v/v) acetonitrile/water risinglinearly to 100% acetonitrile over 25 minutes. The 2 L fermentationyielded about 4 mg of pure trans-3-aza-bicyclo[3.1.0]hexane-2-carboxylicacid rapamycin.

High resolution MS of trans-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acidrapamycin on a Bruker BioApex FTICR mass spectrometer using electrosprayionisation gave a sodiated molecular ion at m/z 934.52776, whichconfirmed the molecular formula to be C₅₁H₇₇NO₁₃.

Biological Activity

The dose response of human lymphoblastoid cell lines 536 was measured.In the experiment shown in FIG. 3 the mean cpm of radiolabelledthymidine incorporated into the untreated controls shows that 0-3 h drugexposure had no appreciable effect on DNA synthesis up to 100 nM ofrapamycin, prolylrapamycin, or 4-hydroxy-prolyl-26-demethoxy-rapamycin.This implies that none of the compounds were toxic to the 536 cell line.After 24 and 48 hours (FIG. 4) the 536 cells showed aconcentration-dependent inhibition of DNA synthesis with an ID50% of inM for rapamycin; and 3 nM for prolylrapamycin.4-hydroxy-prolyl-26-demethoxy-rapamycin was also inhibitory but did notreach 50% at 100 nM. Previous experiments have shown that rapamycin is aprofound inhibitor of G1 progression in the 536 cell line (Metcalfe etal., Oncogene 15, 1635-1642 (1997)). This is also suggested in theseexperiments for the rapamycin analogues, since no significant effect wasfound at 3 h but inhibition was observed once the cell population hadtime to proceed through a complete cell cycle (24 h) and reach the drugarrest point.

1. A process of modifying a gene cluster involved in the biosynthesis ofa polyketide, said gene cluster including a gene (“the precursor gene”)responsible for the production of an enzyme which is responsible for theproduction of a precursor compound which is incorporated into saidpolyketide; said process comprising the step of deleting or inactivatingsaid precursor gene.
 2. A process according to claim 1 wherein saidprocess of deleting or inactivating said precursor gene employsphage-mediated gene replacement.
 3. A process according to claim 1 orclaim 2 wherein the gene cluster is the gene cluster for the productionof rapamycin in S. hygroscopicus and the precursor gene is the rapL genewhose product is responsible for the production of L-pipecolate.
 4. Aprocess for producing a polyketide comprising modifying a gene clusterby the process of claim 1, 2 or 3 and expressing the modified genecluster to produce polyketide synthase enzymes which act in the presenceof a variant precursor compound which is incorporated so that a variantpolyketide is produced.
 5. A process according to claim 4 as appendanton claim 3 wherein the variant precursor compound is selected fromL-proline, L-trans-4-hydroxyproline, L-cis-4-hydroxyproline,L-cis-3-hydroxyproline and trans-3-aza-bicyclo[3,1,0]hexane-2-carboxylicacid.
 6. A compound selected from prolyl-rapamycin, 4-hydroxy-propylrapamycin, 4-hydroxyproplyl-2,6-demethoxy-rapamycin,3-hydroxy-prolyl-rapamycin 3-hydroxy-prolyl-2,6-demethoxy-rapamycin, andtrans-3-aza-bicyclo[3,1,0]hexane-2-carboxylic acid rapamycin.
 7. Apharmaceutical composition comprising a compound of claim
 6. 8. Use of acompound of claim 6 in the manufacture of an immunosuppressantcomposition.
 9. A modified gene cluster as produced by the process ofany of claims 1-3.
 10. A vector containing the gene cluster of claim 9.11. A microorganism containing the gene cluster of claim 9 and capableof expressing it.